Anti-bcma antibodies

ABSTRACT

This invention provides antibodies that recognize the B Cell Maturation Antigen (BCMA) and that bind naïve B cells, plasma cells, and/or memory B cells. The invention further provides methods for depleting naïve B cells, plasma cells, and memory B cells, and for treating B cell-related disorders, including lymphomas and autoimmune diseases.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Mar. 10, 2010, is named08201010.txt, and is 70,657 bytes in size.

This invention relates to antibodies that bind to the B cell surfaceantigen BCMA. The invention also relates to the use of these antibodiesto detect, deplete, and otherwise manipulate various B cell subtypes.

B cells are lymphocytes that play major roles in adaptive humoralimmunity and production of antibodies that specifically recognizeantigens. Three subclasses of B cells are naïve B cells, memory B cells,and plasma cells. The processes of VDJ recombination, in which two orthree segments of DNA are chosen from a genomic library and recombinedto generate a combinatorial array of antibody variable domains, andhypermutation, by which the variable domains encoded by differentlineages of B cells are further varied, result in up to 10⁹ distinct Bcell lineages that produce antibodies with specificity for distincttargets. A B cell is said to be specific for an antigen that binds theantibodies made by that B cell. B cells in general are stimulated byexposure to their specific antigen (Ag). Naïve B cells have not yet beenexposed to their specific antigen. Such exposure (e.g., during aninfection) results in proliferation of B cells and generation of sisterclones. Sister clones can develop into plasma cells, which produce highamounts of antibody. Plasma cells may either be short lived, or maymigrate into bone marrow, where they can persist for an extended periodof time. A sister clone of an Ag-exposed B cell may also develop into amemory B cell that is quiescent until reexposed to the specific antigen.Memory B cells respond rapidly to reexposure to antigen by dividing toproduce both plasma cells and additional memory B cells. Memory B cellsinclude switched memory B cells (CD19⁺CD27^(high)CD38^(low)IgD⁻),unswitched memory B cells (CD19⁺CD27^(high)CD38^(low)IgD⁺), and doublenegative memory B cells (CD19⁺CD27⁻CD38^(low)IgD⁻).

Several significant diseases involve B cells. Malignant transformationof B cells leads to cancers, including some lymphomas such as, forexample, multiple myeloma and Hodgkins' Lymphoma. Some autoimmunediseases, including systemic lupus erythematosus (SLE), also involve Bcells. Both cancer and autoimmune diseases that involve B cells may beconsidered gain of function conditions, in that the B cells overgrowand/or attack parts of the body inappropriately. A possible strategy tocontrol such diseases is to use antibodies that target the pathologicalB cells.

The B cell maturation antigen (BCMA, also known as TNFRSF17 and CD269)is a protein that has been shown to be expressed on the surface ofplasmablasts (i.e., plasma cell precursors) and plasma cells, and isbelieved to stimulate survival. It therefore represents a potentialtarget for B cell-related diseases. BCMA is a member of the TNF receptorfamily and binds the TNF family ligands BAFF and APRIL (reviewed inKalled et al. (2005), Curr Dir Autoimmun 8:206-242). BCMA is a type IIImembrane protein, as it lacks the signal peptide associated with type Imembrane proteins found in most TNF receptor family members.

BCMA RNA has been detected in the spleen, lymph nodes, thymus, adrenalsand liver, and analysis of a number of B cell lines indicated that BCMAmRNA levels increased upon maturation. Human BCMA protein has beendetected on various subtypes of CD38⁺ B cells, particularly plasma cells(Zhang et al. (2005), Int Immunol 17:779-788; Darce et al. (2007), JImmunol 179:7276-7286; Sims et al. (2005), Blood 105:4390-4398; Avery etal. (2003), J Clin Invest 112:286-297). Independent laboratories haveexamined blood and/or tonsil B cell subsets and found that BCMAexpression could not be detected on naïve or memory B cells (Zhang etal. (2005), Int Immunol 17:779-788; Darce et al. (2007), J Immunol179:7276-7286; Chiu et al. (2007), Blood 109:729-739). Attempts todetect BCMA protein on the surface of germinal center B cells have hadinconsistent results (Zhang et al. (2005), Int Immunol 17:779-788; Chiuet al. (2007), Blood 109:729-739).

The mechanism of action of BCMA is not fully understood. Mice that havebeen genetically altered to lack a functional gene for BCMA have normallymphoid organs and cell populations, and a nearly normal functioningimmune system (Xu and Lam (2001), Moll Cell Biol 21:4067-4074; Schiemannet al. (2001), Science 293:2111-2114). The only defect defined to datein these mice is a diminished survival of long-lived bone marrow (BM)plasma cells (O'Connor et al. (2004), J Exp Med 199:91-98). Therefore,it may be that BCMA, at least in the murine system, provides a survivalsignal to BM-resident plasma cells that is either BAFF orAPRIL-mediated, or both. Indeed, signalling through BCMA activates theNF-κB pathway (Hatzoglou et al. (2000), J Immunol 165:1322-1330) whichis involved in B cell survival, proliferation and maturation (Litinskiyet al. (2002) Nat Immunol 3:822-829; Pomerantz and Baltimore (2002) MollCell 10:693-695; Huang et al. (2004) Proc Natl Acad Sci USA101:17789-17794; He et al. (2004) J Immunol 172:3268-3279). Results withmalignant human cells are generally consistent with a link between BCMAand cell survival. Primary multiple myeloma (MM) cells, MM cell lines(Novak et al. (2004) Blood 103:689-694), and Hodgkin and Reed-Sternberg(HRS) cells from Hodgkin lymphomas (Chiu et al. (2007), Blood109:729-739; Novak et al. (2004), Blood 104:2247-2253) have been shownto express BCMA. Addition of BAFF and/or APRIL has further been shown toprovide a survival signal for these malignant cells, although it is notclear that BCMA is predominantly responsible for this effect.

Because different B cell subsets are implicated in different B cellrelated conditions, there exists a need for agents that specificallytarget one or more B cell subsets. The expression of BCMA on the surfaceof some B cells provides a marker by which those cells may bespecifically targeted. To take advantage of BCMA as a marker of one ormore B cell subsets, there is a need for agents that specifically bindto BCMA and for a determination of which B cell subsets are bound bythose BCMA-specific agents. The invention provides antibodies thatspecifically bind to BCMA. The antibodies of the invention may be usedto target one or more of the following B cell subsets: plasma cells,memory B cells, and naïve B cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts binding of anti-human BCMA mAbs on BCMA-transfected CHOcells. Binding of biotin-conjugated anti-human BCMA mAbs, visualizedwith Streptavidin-PE, was measured by flow cytometry on a BCMA-CHOstable cell line (A) and control, non-transfected CHO cells (B). Theshaded area represents staining of cells with an isotype control mAb.

FIG. 2 depicts anti-BCMA binding to B cell subsets. B cell subsets inhuman peripheral blood were assessed by flow cytometry for reactivity toanti-BCMA mAbs. Visualization was as in FIG. 1. The shaded arearepresents staining with an isotype control Ab. B cell subsets wereplasma cells (CD19⁺CD27^(high)CD38^(high)IgD⁻) (A), switched memory Bcells (CD19⁺CD27^(high)CD38^(low)IgD⁻) (B), unswitched memory B cells(CD19⁺CD27^(high)CD38^(low)IgD⁺) (C), double negative memory B cells(CD19⁺CD27⁻CD38^(low)IgD⁻) (D), and naïve B cells (CD19⁺CD27⁻IgD⁺) (E).

FIG. 3 depicts anti-BCMA binding to B cell subsets isolated from healthyand SLE-afflicted individuals. B cell subsets in human peripheral bloodfrom a healthy volunteer and an SLE patient were assessed by flowcytometry for reactivity to the anti-BCMA mAbs C12A3.2 and A7D12.2. Bcell subsets were as in FIG. 2. Visualization was as in FIG. 1.

FIG. 4 depicts flow cytometric staining of human plasma cells within thehuman CD45⁺ splenocyte compartment isolated from HSC/NSG mice. Splenicplasma cells were stained with anti-BCMA antibodies A7D12.2 (left panel;bold line) and C12A3.2 (right panel; bold line) or an isoytpe controlmouse IgG2b or IgG1 Ab, respectively (thin line in both panels).

FIG. 5 depicts flow cytometric staining for plasma cells (PCs) withinthe human CD45⁺ splenocyte compartment isolated from HSC/NSG micetreated with anti-BCMA antibody (chC12A3.2 or chC13F12.1) or human IgG1control. Mice were injected i.p. with anti-BCMA Ab or HIgG1 controltwice weekly for 2 weeks. **p<0.0001; *p=0.0066.

FIG. 6 depicts flow cytometric staining for plasma cells (PCs) withinthe human CD45⁺ splenocyte compartment isolated from HSC/NSG micetreated with anti-BCMA antibody (chC11D5.1 or chA7D12.2) or human IgG1control. Mice were injected i.p. with anti-BCMA Ab or HIgG1 controltwice weekly for 2 weeks.

TABLE 1 Brief Description of the Sequences SEQ ID NO Description ofsequence 1 A7D12.2 mature heavy chain variable domain protein sequence 2A7D12.2 mature light chain variable domain protein sequence 3 C11D5.3mature heavy chain variable domain protein sequence 4 C11D5.3 maturelight chain variable domain protein sequence A 5 C12A3.2 mature heavychain variable domain protein sequence 6 C12A3.2 mature light chainvariable domain protein sequence 7 C13F12.1 mature heavy chain variabledomain protein sequence 8 C13F12.1 mature light chain variable domainprotein sequence 9 BCMA protein sequence 10 huBCMA-huFc (as defined byN-terminal sequence analysis) 11 C11D5.3 mature light chain variabledomain protein sequence B 12 C11D5.3 mature light chain variable domainprotein sequence C 13 chA7D12.2 chimeric mature heavy chain proteinsequence 14 chA7D12.2 chimeric mature light chain protein sequence 15chC11D5.3 chimeric mature heavy chain protein sequence 16 chC11D5.3chimeric mature light chain protein sequence A 17 chC11D5.3 chimericmature light chain protein sequence C 18 chC12A3.2 chimeric mature heavychain protein sequence 19 chC12A3.2 chimeric mature light chain proteinsequence 20 chC13F12.1 chimeric mature heavy chain protein sequence 21chC13F12.1 chimeric mature light chain protein sequence 22 huC11D5.3L1humanized mature light chain variable domain sequence 23 huC11D5.3L2humanized mature light chain variable domain sequence 24 huC11D5.3L3humanized mature light chain variable domain sequence 25 huC11D5.3H0humanized mature heavy chain variable domain sequence 26 huC11D5.3H1humanized mature heavy chain variable domain sequence 27 huC11D5.3H2humanized mature heavy chain variable domain sequence 28 huC11D5.3H3humanized mature heavy chain variable domain sequence 29 huC11D5.3H4humanized mature heavy chain variable domain sequence 30 huC12A3.2L0humanized mature light chain variable domain sequence 31 huC12A3.2L1humanized mature light chain variable domain sequence 32 huC12A3.2L2humanized mature light chain variable domain sequence 33 huC12A3.2L3humanized mature light chain variable domain sequence 34 huC12A3.2H0humanized mature heavy chain variable domain sequence 35 huC12A3.2H1humanized mature heavy chain variable domain sequence 36 huC12A3.2H2humanized mature heavy chain variable domain sequence 37 huC12A3.2H3humanized mature heavy chain variable domain sequence 38 huC12A3.2H4humanized mature heavy chain variable domain sequence 39 huC13F12.1L0humanized mature light chain variable domain sequence 40 huC13F12.1L1humanized mature light chain variable domain sequence 41 huC13F12.1L2humanized mature light chain variable domain sequence 42 huC13F12.1L3humanized mature light chain variable domain sequence 43 huC13F12.1H0humanized mature heavy chain variable domain sequence 44 huC13F12.1H1humanized mature heavy chain variable domain sequence 45 huC13F12.1H2humanized mature heavy chain variable domain sequence 46 huC13F12.1H3humanized mature heavy chain variable domain sequence 47 huC13F12.1H4humanized mature heavy chain variable domain sequence

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention provides antibodies that bind to BCMA and certain epitopesthereof. In some embodiments, the antibodies of the invention bind toone or more subsets of B cells, such as plasma cells, memory B cells,and naïve B cells. The invention also provides methods of depleting Bcells or subclasses of B cells, including plasma cells, memory B cells,and naïve B cells. In one embodiment, the invention provides an isolatedantibody that binds to SEQ ID NO:9 and binds to plasma cells. In oneembodiment, the invention provides an isolated antibody that binds toSEQ ID NO:9 and binds to memory B cells. In another embodiment, theinvention provides an isolated antibody that binds to SEQ ID NO:9 andbinds to naïve B cells.

Certain anti-BCMA mAbs, including clone C4E2.2 (hamster IgG) generatedat Legacy Biogen (6); clone VICKY-1 (rat IgG1) (Alexis Biochemicals,Lausen, Switzerland, also sold as 6D10 by Santa Cruz Biotechnology,Santa Cruz, Calif.); and clone 335004 (rat IgG2a) (R&D Systems, Inc.,Minneapolis, Minn.), are outside the scope of this invention.

A. Antibodies

The invention provides antibodies that bind specifically to SEQ ID NO:9.The invention also provides to antibodies that bind to the surface of Bcells or subclasses thereof, including plasma cells, memory B cells(including switched, unswitched, and double negative), and/or naïve Bcells. The term “antibody” as used herein, includes both full-lengthimmunoglobulins and antibody fragments that bind to the same antigens.The antibodies can be, e.g., a monoclonal, polyclonal, chimeric,humanized, or single chain antibody. In some embodiments, the antibodyfragments are Fab fragments or F(ab′)2 fragments and retain the abilityto specifically bind the protein of SEQ ID NO: 9.

In part, the invention provides the antibodies A7D12.2, C11D5.3,C12A3.2, and C13F12.1. Each of these is a murine monoclonal antibody.A7D12.2 has a murine “miscellaneous” subgroup heavy chain, a heavy chainvariable domain sequence that is SEQ ID NO:1, a subgroup I kappa lightchain, and a light chain variable domain sequence that is SEQ ID NO:2.

C11D5.3 has a subgroup II(A) heavy chain, a heavy chain variable domainsequence that is SEQ ID NO:3, a murine subgroup III kappa light chain,and a light chain variable domain sequence that is selected from SEQ IDNO:4, SEQ ID NO:11, and SEQ ID NO:12. In some embodiments, the lightchain variable domain sequence of C11 D5.3 is SEQ ID NO:12.

C12A3.2 has a subgroup II(A) heavy chain, a heavy chain variable domainsequence that is SEQ ID NO:5, a murine subgroup III kappa light chain,and a light chain variable domain sequence that is SEQ ID NO:6.

C13F12.1 has a subgroup II(A) heavy chain, a heavy chain variable domainsequence that is SEQ ID NO:7, a murine subgroup III kappa light chain,and a light chain variable domain sequence that is SEQ ID NO:8.

Techniques for producing single-chain antibodies specific to the proteinof SEQ ID NO: 9 can be adapted from e.g., those described in U.S. Pat.No. 4,946,778. In addition, methods can be adapted for the constructionof Fab expression libraries (see e.g., Huse et al. (1989) Science246:1275-1281) to allow rapid and effective identification of monoclonalFab fragments with the desired specificity for a BCMA protein orderivatives, fragments, analogs or homologs thereof. Numerous techniquesfor humanizing non-human antibodies are well known in the art. See e.g.,U.S. Pat. Nos. 5,225,539, 6,632,927, or 5,648,237, all of which areincorporated by reference. Antibody fragments that contain the idiotypesto a BCMA protein may be produced by any of a variety of techniques,including, but not limited to: (i) an F(ab′)2 fragment produced bypepsin digestion of an antibody molecule; (ii) an Fab fragment generatedby reducing the disulfide bridges of an F(ab′)2 fragment; (iii) a Fabfragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) Fv fragments.

Additionally, recombinant anti-BCMA antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniques suchas, for example, the methods described in U.S. Pat. No. 7,112,421;Better et al. (1988) Science 240:1041-1043; or Liu et ai. (1987) Proc.Natl. Acad. Sci. USA 84:3439-3443.

Some of the antibodies of the invention are chimeric forms of murinemonoclonal antibodies A7D12.2, C11D5.3, C12A3.2, and C13F12.1. In someembodiments, a chimeric form of A7D12.2 comprises a heavy chaincomprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14. Insome embodiments, a chimeric form of C11 D5.3 comprises a heavy chaincomprising SEQ ID NO:15 and a light chain comprising a sequence selectedfrom SEQ ID NO:16 and SEQ ID NO:17, preferably SEQ ID NO:17. In someembodiments, a chimeric form of C12A3.2 comprises a heavy chaincomprising SEQ ID NO:18 and a light chain comprising SEQ ID NO:19. Insome embodiments, a chimeric form of C13F12.1 comprises a heavy chaincomprising SEQ ID NO:20 and a light chain comprising SEQ ID NO:21.

Some of the antibodies of the invention are humanized forms of murinemonoclonal antibodies A7D12.2, C11D5.3, C12A3.2, and C13F12.1. In someembodiments, a humanized form of C11D5.3 comprises a light chainvariable domain comprising a sequence that is at least 80%, at least85%, at least 90%, at least 95%, or 100% identical to a sequenceselected from SEQ ID NOs 22-24 and a heavy chain variable domainsequence comprising a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or 100% identical to a sequence selected fromSEQ ID NOs 25-29. In some embodiments, a humanized form of C12A3.2comprises a light chain variable domain comprising a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, or 100% identicalto a sequence selected from SEQ ID NOs 30-33 and a heavy chain variabledomain sequence comprising a sequence that is at least 80%, at least85%, at least 90%, at least 95%, or 100% identical to a sequenceselected from SEQ ID NOs 34-38. In some embodiments, a humanized form ofC13F12.1 comprises a light chain variable domain comprising a sequencethat is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to a sequence selected from SEQ ID NOs 39-42 and a heavy chainvariable domain sequence comprising a sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, or 100% identical to a sequenceselected from SEQ ID NOs 43-47.

B. Antibody Variable Domain Sequence

The antibodies of the invention may comprise the heavy chain variabledomain sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or

SEQ ID NO:7. The heavy chain variable domain sequences may consistessentially of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

The antibodies of the invention may comprise the light chain variabledomain sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:11, or SEQ ID NO:12. The light chain variable domain sequencesmay consist essentially of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:11, or SEQ ID NO:12.

The invention also provides a variable domain sequence comprising asequence that is at least 80%, at least 85%, at least 90%, or at least95% identical to a sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, and SEQ ID NO:7. The invention also provides a variable domainsequence comprising a sequence that is at least 80%, at least 85%, atleast 90%, or at least 95% identical to a sequence selected from SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, and SEQ IDNO:12. The invention also provides antibodies comprising a heavy chainvariable domain sequence that is at least 80%, at least 85%, at least90%, or at least 95% identical to SEQ ID NO:1 and a light chain variabledomain sequence that is at least 80%, at least 85%, at least 90%, or atleast 95% identical to SEQ ID NO:2. The invention includes antibodiescomprising a heavy chain variable domain sequence that is at least 80%,at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3 anda light chain variable domain sequence that is at least 80%, at least85%, at least 90%, or at least 95% identical to SEQ ID NO: 4, SEQ IDNO:11, or SEQ ID NO:12. The invention includes antibodies comprising aheavy chain variable domain sequence that is at least 80%, at least 85%,at least 90%, or at least 95% identical to SEQ ID NO:5 and a light chainvariable domain sequence that is at least 80%, at least 85%, at least90%, or at least 95% identical to SEQ ID NO:6. The invention includesantibodies comprising a heavy chain variable domain sequence that is atleast 80%, at least 85%, at least 90%, or at least 95% identical to SEQID NO:7 and a light chain variable domain sequence that is at least 80%,at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:8.

The invention also provides antibodies with particular complementaritydetermining regions (CDR). Table 2 defines the amino acid coordinates ofCDR1, CDR2, and CDR3 of SEQ ID NOs:1 through 8, 11, and 12.

TABLE 2 CDR Amino Acid Coordinates SEQ ID NO Description CDR1 CDR2 CDR31 A7D12.2 V_(H) 31-35 50-66 99-111 2 A7D12.2 V_(L) 24-34 50-56 89-97  3C11D5.3 V_(H) 31-35 50-66 99-106 4 C11D5.3 V_(L) A 24-38 54-60 93-101 5C12A3.2 V_(H) 31-35 50-66 99-106 6 C12A3.2 V_(L) 24-38 54-60 93-101 7C13F12.1 V_(H) 31-35 50-66 99-106 8 C13F12.1 V_(L) 24-38 54-60 93-101 11C11D5.3 V_(L) B 24-38 54-60 93-101 12 C11D5.3 V_(L) C 24-38 54-60 93-101

CDRs are designated using the Kabat definitions (Johnson and Wu (2000),Nucleic Acids Res 28:214-218). As used herein, the “corresponding CDR”means the CDR in the most similar position within the variable domainamino acid sequence.

The heavy chain variable domain of antibodies of the invention maycomprise CDRs such that one, two, or three of the CDRs are identical tothe corresponding CDRs of SEQ ID NO:1; identical to the correspondingCDRs of SEQ ID NO:3; identical to the corresponding CDRs of SEQ ID NO:5;or identical to the corresponding CDRs of SEQ ID NO:7. The light chainvariable domain of antibodies of the invention may comprise CDRs suchthat one, two, or three of the CDRs are identical to the correspondingCDRs of SEQ ID NO:2; identical to the corresponding CDRs of SEQ ID NO:4;identical to the corresponding CDRs of SEQ ID NO:6; identical to thecorresponding CDRs of SEQ ID NO:8; identical to the corresponding CDRsof SEQ ID NO:11; or identical to the corresponding CDRs of SEQ ID NO:12.

The heavy chain variable domain may comprise CDRs identical to each ofthe corresponding CDRs of one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,or SEQ ID NO:7 except that one or more amino acid substitutions havebeen made in said CDR regions. In certain embodiments, CDRs of the heavychain variable domain may have up to a total of 12 amino acidsubstitutions relative to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQID NO:7. In other embodiments, the heavy chain variable domain CDRs ofthe antibodies of the invention may have up to 10, up to 8, up to 5, orup to 3 substitutions relative to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,or SEQ ID NO:7. In some embodiments, the heavy chain variable domainCDRs of the antibodies of the invention are at least 80%, at least 85%,at least 90%, or at least 95% identical to the heavy chain variabledomain CDRs of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

In some embodiments, CDR2 of SEQ ID NO:7 is replaced by CDR2 (i.e.,amino acids 50-66) of SEQ ID NO:46. For example, the heavy chainvariable domain of an antibody of the invention may comprise CDR1 andCDR3 of SEQ ID NO:7 and CDR2 of SEQ ID NO:46. The heavy chain variabledomain of an antibody of the invention may also comprise CDR1, CDR2, andCDR3 regions that are together at least 80%, at least 85%, at least 90%,or at least 95% identical to CDR1 and CDR3 of SEQ ID NO:7 and CDR2 ofSEQ ID NO:46.

The light chain variable domain may comprise CDRs identical to thecorresponding CDRs of one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:11, or SEQ ID NO:12, except for one or more aminoacid substitutions in said CDR regions. In certain embodiments, theantibodies of the invention comprise CDRs that are identical to thecorresponding CDRs of one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:11, or SEQ ID NO:12, except for up to 12, up to 10,up to 8, up to 5, or up to 3 amino acid substitutions in said CDRregions. In some embodiments, the light chain variable domain CDRs ofthe antibodies of the invention are at least 80%, at least 85%, at least90%, or at least 95% identical to the light chain variable domain CDRsof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, orSEQ ID NO:12.

In some embodiments, the substitutions in the CDR regions areconservative substitutions.

C. Epitopes; Antibody Binding Specificity

The invention also provides antibodies that bind particular epitopes.Whether a pair of antibodies binds the same epitope is determined basedon cross-blocking experiments, as described in Example 4. Cross-blockingprofiles are defined for seven antibodies in Table 3. For the purposesof this disclosure, two antibodies are considered to bind to the sameepitope if each one reduces the other's binding to BCMA (i.e., theymutually cross-block) by at least 90% according to the proceduredescribed in Example 4. Similarly, antibodies that do not reduce eachother's binding by at least 90% as described in Example 4 are consideredto bind to distinct epitopes. The cross-blocking profiles of antibodieswith certain variable domains are listed in Table 3. Pairs of antibodiesthat bind to distinct epitopes (as defined above) are noted with a “d.”

TABLE 3 Cross-Blocking Profiles Heavy chain Light chain var. domain var.domain Distinct (d) from epitope bound by: SEQ ID NO SEQ ID NO ExampleC12A3.2 C11D5.3 C13F12.1 335004 C4E2 A7D12.2 VICKY-1 1 2 A7D12.2 d d d dd — d 3 12 C11D5.3 — — — d d d d 5 6 C12A3.2 — — — d d d d 7 8 C13F12.1— — — d d d d

The invention further encompasses antibodies that bind to the sameepitope as antibodies A7D12.2, C11D5.3, C12A3.2, or C13F12.1. Theinvention also provides antibodies that have cross-blocking profilesthat match the profiles of A7D12.2, C11D5.3, C12A3.2, or C13F12.1. Forexample, following the definitions provided above, an antibody that hasthe same profile as C11D5.3 binds to the same epitope as compared toC12A3.2 and C13F12.1 but binds to a distinct epitope as compared toA7D12.2, 335004, C4E2, and Vicky-1. In some embodiments, the antibodiesof the invention mutually cross-block one or more of A7D12.2, C11D5.3,C12A3.2, or C13F12.1 from binding the protein of SEQ ID NO: 9 by atleast 80%, 85%, 90%, or 95%. The extent of cross-blocking is measuredaccording to the procedure described in Example 4.

In some embodiments, the antibodies and antibody fragments of theinvention bind to the extracellular domain of BCMA. In particularembodiments, the antibodies bind to amino acids 1-52, 1-51, 1-41, or8-41 of SEQ ID NO:9.

The invention also provides anti-BCMA antibodies that bind to one ormore particular types of cell. The antibodies or antibody fragments ofthe invention may bind one or more of the following: plasma cells,memory B cells, naïve B cells, or cells that express BCMA (SEQ ID NO:9),a protein similar thereto, the extracellular domain thereof, or apolypeptide similar to the extracellular domain thereof.

D. Methods

The invention provides methods of depleting various types of cells. Themethods comprise administering the antibodies of the invention, asdescribed above. Types of cells that may be depleted by the methods ofthe invention include, without limitation, plasma cells, naïve B cells,memory B cells (including switched, unswitched, and double negative),lymphoma cells derived from B cells, and cells that express BCMA, aprotein similar thereto, the extracellular domain thereof, or apolypeptide similar to the extracellular domain thereof. A cell may bein more than one of the foregoing categories. For an example ofantibody-mediated cell depletion methods, see “Depletion of B Cells InVivo by a Chimeric Mouse Human Monoclonal Antibody to CD20”, Mitchell E.Reff, Blood, vol. 83, pp. 435-445, Jan. 15, 1994.

In some embodiments, treatment with an antibody of the invention reducesthe number of one or more of the above-listed cell types by at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, and least85%, at least 90%, or at least 95%. In some embodiments, treatment withan antibody of the invention reduces the number of plasma cells by atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%. In some embodiments, treatment with anantibody of the invention reduces the number of switched memory B cellsby at least 25%, at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, or at least 60%. In some embodiments, treatment with anantibody of the invention reduces the number of unswitched memory Bcells by at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, or at least 60%. In some embodiments, treatmentwith an antibody of the invention reduces the number of double negativememory B cells by at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, or at least 60%. In some embodiments,treatment with an antibody of the invention reduces the number of naiveB cells by at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, or at least 60%.

The invention also provides methods of reducing serum immunoglobulinlevels comprising administering an antibody of the invention. In someembodiments, such methods reduce serum IgM levels. In particularembodiments, treatment with an antibody of the invention reduces serumIgM levels by at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, or at least 65%. Insome embodiments, such methods reduce serum IgG levels. In someembodiments, such methods reduce the levels of one or both of IgG2 andIgG3. In some embodiments, such methods reduce the levels of IgG2 by atleast 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 65%, or at least 70%. In some embodiments, suchmethods reduce the levels of IgG3 by at least 25%, at least 30%, atleast 35%, at least 40%, at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, or at least 80%. In some embodiments, suchmethods reduce IgG2, IgG3, and IgM levels.

The invention also provides methods of reducing the level of at leastone autoantibody comprising administering an antibody of the invention.In some embodiments, such methods reduce the level of one or moreautoantibodies by at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, and least 85%, at least 90%, or at least 95%.

In a still further aspect, the invention provides methods of treating orpreventing or delaying a B-cell mediated condition disorder. The methodincludes administering to a subject in which such treatment orprevention or delay is desired, an antibody of the invention in anamount sufficient to treat, prevent, or delay a tumorigenic orimmunoregulatory condition in the subject. In some embodiments, thesubject is a human. In other embodiments, the subject is a non-humanmammal. In some embodiments, administration of the antibody of theinvention blocks BCMA-mediated signalling in the subject, which mayresult in one or more of cell death, inhibition, reduction, or cessationof cell proliferation.

In some embodiments, the antibodies or antibody fragments of theinvention use BCMA to “target” B cell lymphomas. In essence, suchtargeting can be generalized as follows: antibodies or antibodyfragments of the invention specific to the BCMA surface antigen of Bcells are, e.g., injected into a subject and specifically bind to theBCMA cell surface antigen of (ostensibly) both normal and malignant Bcells; this binding leads to the destruction and/or depletion ofneoplastic B cells. Additionally, chemical agents or radioactive labelshaving the potential to destroy cancer cells and/or tumors can beconjugated to the antibodies or antibody fragments of the invention suchthat the agent is specifically “delivered” to the targeted B cells, suchas, e.g., neoplastic B cells. In some embodiments, the methods of theinvention comprise administering an antibody or antibody fragment thatis not conjugated to a chemical agent or radioactive label. In someembodiments, the methods of the invention comprise administering anantibody or antibody fragment that is not conjugated to a cytotoxicagent.

B cell-related disorders include, without limitation, autoimmunediseases involving inappropriate B cell activity and B cell lymphomas. Bcell lymphomas include, without limitation, multiple myeloma,plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, smallnon-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadicBurkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associatedlymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, spleniclymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed celllymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma,pulmonary B cell angiocentric lymphoma, and small lymphocytic lymphoma.The antibodies or antibody fragments of the invention may also be usedto treat cancers in which the cancer cells express BCMA. The Bcell-related disorders additionally include B cell proliferations ofuncertain malignant potential, such as, for example, lymphomatoidgranulomatosis and post-transplant lymphoproliferative disorder.

The conditions diagnosed, treated, prevented or delayed using theantibodies or antibody fragments of the invention can additionally be animmunoregulatory disorder. These disorders include those that areautoimmune in nature such as, for example, systemic lupus erythematosus,rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia,idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas'disease, Grave's disease, Wegener's granulomatosis, poly-arteritisnodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiplesclerosis, anti-phospholipid syndrome, ANCA associated vasculitis,Goodpasture's disease, Kawasaki disease, and rapidly progressiveglomerulonephritis. The antibodies or antibody fragments of theinvention may also have application in plasma cell disorders such asheavy-chain disease, primary or immunocyte-associated amyloidosis, andmonoclonal gammopathy of undetermined significance (MGUS).

Compositions and methods of treatment using the antibodies or antibodyfragments of the invention can be used with any condition associatedwith undesired BCMA-expressing cell proliferation.

The antibodies of the invention may also be administered in conjunctionwith antibody C2B8 of U.S. Pat. No. 5,736,137, also known as RITUXAN™.In some embodiments, such combined administration depletes or inhibitsthe proliferation of multiple B cell subtypes.

The invention further provides for the use of the antibodies of theinvention to assay B cell phenotypes, such as determination of thepresence, absence, or amount of a marker on the surface of a B cell or Bcell subtype. For example, the antibodies of the invention may be usedto measure the presence of a marker associated with SLE or another Bcell-related condition on the surface of naïve B cells, memory B cells,IgD+ memory B cells, IgD− memory B cells, or double negative memory Bcells.

E. Pharmaceutical Compositions

The antibodies of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise antibodies and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, diluents,and the like, compatible with pharmaceutical administration. Suitablecarriers are described in the most recent edition of Remington'sPharmaceutical Sciences, a standard reference text in the field, whichis incorporated herein by reference. Preferred examples of such carriersor diluents include, but are not limited to, water, saline, finger'ssolutions, dextrose solution, and 5% human serum albumin. Liposomes andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the antibodies, use thereof in the compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition comprising antibodies of the invention isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufferssuch as acetates, citrates or phosphates, and agents for the adjustmentof tonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating antibodiesof the invention in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the antibodies into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the antibodies plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one embodiment, the antibodies are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. Additionally,the antibodies or antibody fragments of the invention may be used totarget liposomal suspensions to B cells or subclasses thereof to whichthe particular antibody binds. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subject to be treated; each unit containing a predetermined quantityof antibody calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions comprising an antibody of the inventioncan be included in a container, pack, or dispenser together withinstructions for administration.

F. Listing of Exemplary Embodiments:

-   1. An isolated antibody that binds to SEQ ID NO:9 and binds to    memory B cells.-   2. The antibody of embodiment 1, wherein the antibody also binds to    plasma cells.-   3. The antibody of embodiment 1, wherein the antibody also binds to    naïve B cells.-   4. The antibody of embodiment 1, wherein the antibody also binds to    naïve B cells and plasma cells.-   5. A method of depleting naïve B cells, memory B cells, and plasma    cells, comprising administering an antibody that binds to SEQ ID    NO:9 and binds to naïve B cells, memory B cells, and plasma cells.-   6. A method of depleting memory B cells, comprising administering an    antibody that binds to SEQ ID NO:9 and binds to memory B cells.-   7. An isolated antibody that binds to SEQ ID NO:9 and binds to naïve    B cells.-   8. The antibody of embodiment 7, wherein the antibody binds to    plasma cells.-   9. A method of depleting naïve B cells, comprising administering an    antibody that binds to SEQ ID NO:9 and binds to naïve B cells.-   10. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises at least one CDR chosen from    CDR1, CDR2, or CDR3 of a protein sequence selected from the group    consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID    NO:7.-   11. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of a protein sequence    selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ    ID NO:5, and SEQ ID NO:7.-   12. The antibody of embodiment 10, wherein the antibody comprises    the CDRs at CDR1, CDR2, and CDR3 of a protein sequence selected from    the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and    SEQ ID NO:7.-   13. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 1, and the    antibody further comprises a variable domain comprising at least one    of a CDR1 region identical to amino acids 24-34 of SEQ ID NO:2, a    CDR2 region identical to amino acids 50-56 of SEQ ID NO:2, or a CDR3    region identical to amino acids 89-97 of SEQ ID NO:2.-   14. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 3, and the    antibody further comprises a variable domain comprising at least one    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 12, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 12, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 12.-   15. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 5, and the    antibody further comprises a variable domain comprising at least one    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 6, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 6, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 6.-   16. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 7, and the    antibody further comprises a variable domain comprising at least one    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 8, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 8, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 8.-   17. The antibody of embodiment 10, wherein the heavy chain variable    domain comprises SEQ ID NO:1 and the light chain variable domain    comprises SEQ ID NO:2.-   18. The antibody of embodiment 10, wherein the heavy chain variable    domain comprises SEQ ID NO:3 and the light chain variable domain    comprises SEQ ID NO:12. 19. The antibody of embodiment 10, wherein    the heavy chain variable domain comprises SEQ ID NO:5 and the light    chain variable domain comprises SEQ ID NO:6.-   20. The antibody of embodiment 10, wherein the heavy chain variable    domain comprises SEQ ID NO:7 and the light chain variable domain    comprises SEQ ID NO:8.-   21. An isolated antibody that binds the same epitope as the antibody    of embodiment 17.-   22. An isolated antibody that binds the same epitope as the antibody    of any of embodiments 18-20.-   23. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises:    -   a. a variable domain comprising CDR1, CDR2, and CDR3 regions        identical to the CDR1, CDR2, and CDR3 of a sequence which is        chosen from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID        NO:7; or    -   b. a variant of the variable domain of part (a) that is        otherwise identical to said variable domain except for up to a        total of 10 amino acid substitutions in said CDR regions.-   24. The antibody of embodiment 53, wherein the sequence is SEQ ID    NO:1 and the antibody further comprises:    -   c. a variable domain comprising a CDR1 region identical to amino        acids 24-34 of SEQ ID NO:2, a CDR2 region identical to amino        acids 50-56 of SEQ ID NO:2, and a CDR3 region identical to amino        acids 89-97 of SEQ ID NO:2; or    -   d. a variant of the variable domain of part (c) that is        otherwise identical to said variable domain except for up to a        total of 10 amino acid substitutions in said CDR regions.-   25. The antibody of embodiment 53, wherein the sequence is SEQ ID    NO:3 and the antibody further comprises:    -   c. a variable domain comprising a CDR1 region identical to amino        acids 24-38 of SEQ ID NO:12, a CDR2 region identical to amino        acids 54-60 of SEQ ID NO:12, and a CDR3 region identical to        amino acids 93-101 of SEQ ID NO:12; or    -   d. a variant of the variable domain of part (c) that is        otherwise identical to said variable domain except for up to a        total of 10 amino acid substitutions in said CDR regions.-   26. The antibody of embodiment 53, wherein the sequence is SEQ ID    NO:5 and the antibody further comprises:    -   c. a variable domain comprising a CDR1 region identical to amino        acids 24-38 of SEQ ID NO:6, a CDR2 region identical to amino        acids 54-60 of SEQ ID NO:6, and a CDR3 region identical to amino        acids 93-101 of SEQ ID NO:6; or    -   d. a variant of the variable domain of part (c) that is        otherwise identical to said variable domain except for up to a        total of 10 amino acid substitutions in said CDR regions.-   27. The antibody of embodiment 53, wherein the sequence is SEQ ID    NO:7 and the antibody further comprises:    -   c. a variable domain comprising a CDR1 region identical to amino        acids 24-38 of SEQ ID NO:8, a CDR2 region identical to amino        acids 54-60 of SEQ ID NO:8, and a CDR3 region identical to amino        acids 93-101 of SEQ ID NO:8; or    -   d. a variant of the variable domain of part (c) that is        otherwise identical to said variable domain except for up to a        total of 10 amino acid substitutions in said CDR regions.-   28. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises at least one CDR chosen from    CDR1, CDR2, or CDR3 of a protein sequence selected from the group    consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8;    SEQ ID NO:11; and SEQ ID NO:12.-   29. The antibody of embodiment 28, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of a protein sequence    selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ    ID NO:6, and SEQ ID NO:8.-   30. The antibody of embodiment 28, wherein the antibody comprises    the CDRs at CDR1, CDR2, and CDR3 of a protein sequence selected from    the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:6, or    SEQ ID NO:8.-   31. The antibody of any of embodiments 1-4, 7, 8, 10-21, or 23-30,    wherein the antibody is a chimeric, humanized, or single chain    antibody.-   32. A hybridoma that produces the antibody of any of embodiments    1-4, 7, 8, 10-21, or 23-30.-   33. A pharmaceutical composition comprising the antibody of any of    embodiments 1-4, 7, 8, 10-21, or 23-30 and a pharmaceutically    acceptable carrier.-   34. A polypeptide that binds to SEQ ID NO:9 and comprises the    antigen binding portion, Fab fragment, or F(ab′)2 fragment of the    antibody of any of embodiments 1-4, 7, 8, 10-21, or 23-30.-   35. A hybridoma that produces the antigen binding portion, Fab    fragment, or F(ab′)2 fragment of embodiment 33.-   36. A pharmaceutical composition comprising the antigen binding    portion, Fab fragment, or F(ab′)2 fragment of embodiment 33 and a    pharmaceutically acceptable carrier.-   37. A method of depleting plasma cells, comprising administering the    antibody of any of embodiments 1-4, 8, or 10-31.-   38. A method of treating a B cell-related disorder, comprising    administering the antibody of any one of embodiments 1-4, 8, or    10-31.-   39. The method of embodiment 38, wherein the B-cell related disorder    is plasmacytoma, Hodgkins' lymphoma, follicular lymphomas, small    non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic    Burkitt's lymphoma, marginal zone lymphoma, extranodal    mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell    lymphoma, splenic lymphoma, mantle cell lymphoma, large cell    lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma,    primary mediastinal B cell lymphoma, pulmonary B cell angiocentric    lymphoma, small lymphocytic lymphoma, B cell proliferations of    uncertain malignant potential, lymphomatoid granulomatosis,    post-transplant lymphoproliferative disorder, an immunoregulatory    disorder, rheumatoid arthritis, myasthenia gravis, idiopathic    thrombocytopenia purpura, anti-phospholipid syndrome, Chagas'    disease, Grave's disease, Wegener's granulomatosis, poly-arteritis    nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma,    multiple sclerosis, anti-phospholipid syndrome, ANCA associated    vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune    hemolytic anemia, and rapidly progressive glomerulonephritis,    heavy-chain disease, primary or immunocyte-associated amyloidosis,    or monoclonal gammopathy of undetermined significance.-   40. The method of embodiment 38, wherein the B cell-related disorder    is a B cell malignancy.-   41. The method of embodiment 38, wherein the B cell-related disorder    is a plasma cell malignancy.-   42. The method of embodiment 41, wherein the plasma cell malignancy    is multiple myeloma.-   43. The method of embodiment 38, wherein the B cell-related disorder    is an autoimmune disease.-   44. The method of embodiment 43, wherein the autoimmune disease is    systemic lupus erythematosus.-   45. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 1, and the    antibody further comprises a variable domain comprising at least two    of a CDR1 region identical to amino acids 24-34 of SEQ ID NO:2, a    CDR2 region identical to amino acids 50-56 of SEQ ID NO:2, or a CDR3    region identical to amino acids 89-97 of SEQ ID NO:2.-   46. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 1, and the    antibody further comprises a variable domain comprising a CDR1    region identical to amino acids 24-34 of SEQ ID NO:2, a CDR2 region    identical to amino acids 50-56 of SEQ ID NO:2, and a CDR3 region    identical to amino acids 89-97 of SEQ ID NO:2.-   47. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 3, and the    antibody further comprises a variable domain comprising at least two    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 12, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 12, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 12.-   48. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 3, and the    antibody further comprises a variable domain comprising a CDR1    region identical to amino acids 24-38 of SEQ ID NO: 12, a CDR2    region identical to amino acids 54-60 of SEQ ID NO: 12, and a CDR3    region identical to amino acids 93-101 of SEQ ID NO: 12.-   49. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 5, and the    antibody further comprises a variable domain comprising at least two    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 6, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 6, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 6.-   50. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 5, and the    antibody further comprises a variable domain comprising a CDR1    region identical to amino acids 24-38 of SEQ ID NO: 6, a CDR2 region    identical to amino acids 54-60 of SEQ ID NO: 6, and a CDR3 region    identical to amino acids 93-101 of SEQ ID NO: 6.-   51. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 7, and the    antibody further comprises a variable domain comprising at least two    of a CDR1 region identical to amino acids 24-38 of SEQ ID NO: 8, a    CDR2 region identical to amino acids 54-60 of SEQ ID NO: 8, or a    CDR3 region identical to amino acids 93-101 of SEQ ID NO: 8.-   52. The antibody of embodiment 10, wherein the at least one CDR    chosen from CDR1, CDR2, or CDR3 is from SEQ ID NO: 7, and the    antibody further comprises a variable domain comprising a CDR1    region identical to amino acids 24-38 of SEQ ID NO: 8, a CDR2 region    identical to amino acids 54-60 of SEQ ID NO: 8, and a CDR3 region    identical to amino acids 93-101 of SEQ ID NO: 8.-   53. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:1 and at    least one CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:2.-   54. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:3 and at    least one CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:12.-   55. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:5 and at    least one CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:6.-   56. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:7 and at    least one CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:8.-   57. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, and CDR3 of SEQ ID NO:1 and at least one    CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:2.-   58. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:3 and at least one    CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:12.-   59. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:5 and at least one    CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:6.-   60. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:7 and at least one    CDR chosen from CDR1, CDR2, or CDR3 of SEQ ID No:8.-   61. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:1 and at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:2.-   62. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:3 and at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:12.-   63. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:5 and at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:6.-   64. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:7 and at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:8.-   65. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, and CDR3 of SEQ ID NO:1 and at least two    CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:2.-   66. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:3 and at least two    CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:12.-   67. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:5 and at least two    CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:6.-   68. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:7 and at least two    CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID No:8.-   69. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:1 and    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID No:2.-   70. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:3 and    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID No:12.-   71. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:5 and    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID No:6.-   72. The antibody of embodiment 10, wherein the antibody comprises at    least two CDRs chosen from CDR1, CDR2, or CDR3 of SEQ ID NO:7 and    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID No:8.-   73. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, and CDR3 of SEQ ID NO:1 and CDRs that are    CDR1, CDR2, or CDR3 of SEQ ID No:2.-   74. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:3 and CDRs that are    CDR1, CDR2, or CDR3 of SEQ ID No:12.-   75. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:5 and CDRs that are    CDR1, CDR2, or CDR3 of SEQ ID No:6.-   76. The antibody of embodiment 10, wherein the antibody comprises    CDRs that are CDR1, CDR2, or CDR3 of SEQ ID NO:7 and CDRs that are    CDR1, CDR2, or CDR3 of SEQ ID No:8.-   77. An isolated antibody that mutually cross-blocks the antibody of    embodiment 17 by at least 80%.-   78. An isolated antibody that mutually cross-blocks at least one of    the antibodies of embodiments 18-20 by at least 80%.-   79. An isolated antibody that binds to the same epitope as the    antibody of embodiment 18.-   80. An isolated antibody that binds to the same epitope as the    antibody of embodiment 19.-   81. An isolated antibody that binds to the same epitope as the    antibody of embodiment 20.-   82. A method of reducing the level of at least one autoantibody,    comprising administering the antibody of any of embodiments 1-4, 8,    and 10-31.-   83. A method of reducing serum IgM level, comprising administering    the antibody of any of embodiments 1-4, 8, and 10-31.-   84. A method of reducing serum IgG level, comprising administering    the antibody of any of embodiments 1-4, 8, and 10-31.-   85. A method of reducing serum IgG2 level, comprising administering    the antibody of any of embodiments 1-4, 8, and 10-31.-   86. A method of reducing serum IgG3 level, comprising administering    the antibody of any of embodiments 1-4, 8, and 10-31.-   87. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises a variable domain comprising    CDR1, CDR2, and CDR3 regions that are together at least 95%    identical to the CDR1, CDR2, and CDR3 regions of a sequence which is    chosen from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.-   88. The antibody of embodiment 87, wherein the sequence is SEQ ID    NO:1 and the antibody further comprises a variable domain comprising    CDR1, CDR2, and CDR3 regions that are together at least 95%    identical to the CDR1, CDR2, and CDR3 regions of SEQ ID NO:2.-   89. The antibody of embodiment 87, wherein the sequence is SEQ ID    NO:3 and the antibody further comprises a variable domain comprising    CDR1, CDR2, and CDR3 regions that are together at least 95%    identical to the CDR1, CDR2, and CDR3 regions of SEQ ID NO:12.-   90. The antibody of embodiment 87, wherein the sequence is SEQ ID    NO:5 and the antibody further comprises a variable domain comprising    CDR1, CDR2, and CDR3 regions that are together at least 95%    identical to the CDR1, CDR2, and CDR3 regions of SEQ ID NO:6.-   91. The antibody of embodiment 87, wherein the sequence is SEQ ID    NO:7 and the antibody further comprises a variable domain comprising    CDR1, CDR2, and CDR3 regions that are together at least 95%    identical to the CDR1, CDR2, and CDR3 regions of SEQ ID NO:8.-   92. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises a light chain variable domain    comprising a sequence that is at least 95% identical to a sequence    selected from SEQ ID NOs 22-24 and a heavy chain variable domain    sequence comprising a sequence that is at least 95% identical to a    sequence selected from SEQ ID NOs 25-29.-   93. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises a light chain variable domain    comprising a sequence that is at least 95% identical to a sequence    selected from SEQ ID NOs 30-33 and a heavy chain variable domain    sequence comprising a sequence that is at least 95% identical to a    sequence selected from SEQ ID NOs 34-38.-   94. An isolated antibody that binds to the polypeptide of SEQ ID    NO:9, wherein the antibody comprises a light chain variable domain    comprising a sequence that is at least 95% identical to a sequence    selected from SEQ ID NOs 39-42 and a heavy chain variable domain    sequence comprising a sequence that is at least 95% identical to a    sequence selected from SEQ ID NOs 43-47.-   95. The method of embodiment 39, wherein the B-cell related disorder    is rheumatoid arthritis.-   96. The method of embodiment 39, wherein the B-cell related disorder    is idiopathic thrombocytopenia purpura, or myasthenia gravis, or    autoimmune hemolytic anemia.-   97. The method of embodiment 37, further comprising administering    RITUXAN™.-   98. The method of embodiment 38, further comprising administering    RITUXAN™.

EXAMPLE 1 Generation and Biotin Conjugation of Anti-Human BCMAMonoclonal Antibodies

Anti-BCMA monoclonal antibodies (mAbs) were generated by immunizingfemale RBF mice with BCMA-Fc/KLH conjugate protein i.p. in CFA, followedby additional immunizations at regular intervals with IFA, except thatthe last boost used RIBI instead of IFA, prior to splenocyte fusion tothe FL653 myeloma cell line after the method of Harlow and Lane (1998),Using Antibodies: A Laboratory Manual: Portable Protocol No. I, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. Briefly, splenocytesisolated from a mouse 3 days after the final boost were washed twice andmixed in a 7:1 ratio with twice-washed log phase FL653 myeloma cells.The cell mixture was split four ways, pelleted, and incubated in 37° C.PEG for 1 min during which time cells were gently resuspended, followedby careful addition of 10 ml ice-cold DMEM. Cells were mixed, pelleted,and resuspended in AAT hybridoma growth selection media. Cellsupernatants were screened for BCMA-specific reactivity by ELISA andflow cytometry. Clones that scored positive for BCMA and negative forFc-specificity in an ELISA format, positive on BCMA-transfected cells,and negative on mock-transfected cells were expanded and subcloned. FourBCMA-specific clones were selected for further evaluation: C11D5.3(IgG1), C12A3.2 (IgG1), C13F12.1 (IgG1) and A7D12.2 (IgG2b). Anti-BCMAmAbs were biotin-conjugated for use in ELISA and FACS experimentsdescribed below using a kit according to the manufacturer'srecommendations (Molecular Probes, Eugene, Oreg.).

EXAMPLE 2 Cloning of Murine Anti-Human BCMA mAb Variable Regions

Total cellular RNA from murine hybridoma cells was prepared using aQiagen RNeasy mini kit following the manufacturer's recommendedprotocol. cDNAs encoding the variable regions of the heavy and lightchains were cloned by RT-PCR from total cellular RNA, using randomhexamers for priming of first strand cDNA. For PCR amplification of themurine immunoglobulin variable domains with intact signal sequences, acocktail of degenerate forward primers hybridizing to multiple murineimmunoglobulin gene family signal sequences and a single back primerspecific for the 5′ end of the murine constant domain were used. PCR wasperformed using Clontech Advantage 2 Polymerase mix following themanufacturer's recommended protocol. The PCR products were gel-purifiedand subcloned into Invitrogen's pCR2.1TOPO vector using their TOPOcloning kit following the manufacturer's recommended protocol. Insertsfrom multiple independent subclones were sequenced to establish aconsensus sequence. Deduced mature immunoglobulin N-termini wereconsistent with those determined by Edman degradation from thehybridoma. Assignment to specific subgroups was based upon BLASTanalysis using consensus immunoglobulin variable domain sequences fromthe Kabat database (Johnson and Wu (2000), Nucleic Acids Res28:214-218). CDRs were designated using the Kabat definitions (Johnsonand Wu (2000), Nucleic Acids Res 28:214-218).

EXAMPLE 3 Stable BCMA-Expressing CHO Cell Line Development andAssessment of Anti-Human BCMA mAbs

To validate specific binding to BCMA, anti-BCMA mAbs were screened on astable BCMA-expressing CHO cell line. The stable BCMA-expressing CHOcell line was generated using a previously described method (Brezinskyet al. (2003), J Immunol Methods 277:141-155). Briefly, approximately1.5 million dihydrofolate reductase (DHFR) deficient DG44 Chinesehamster ovary (CHO) cells were transfected with 4 μg PV90 plasmid DNAcontaining the human BCMA gene using Fugene 6 Transfection Reagent(Roche, Indianapolis, Ind.). Following transfection, the cells werecultured in 6-well culture dishes. Twenty-four hours post-transfection,the growth medium was changed to alpha minus MEM (Gibco, Rockville,Md.), 10% dialyzed serum (Hyclone, Logan, Utah), and 2 mM L-glutamine(Gibco, Rockville, Md.), and cells were pooled and split into three T-75tissue culture flasks and allowed to grow to confluence. Seven dayspost-transfection, the cells were pooled again and split into five T225tissue culture flasks and allowed to grow to confluence.

Fourteen days post-transfection, the cells were incubated with theC4E2.2 Ab (6) and sorted for BCMA+ cells. These cells were grown inculture and sorted a second time, with positive cells sorted into96-well plates. Clones were expanded and assessed for BCMA expressionusing clone C4E2.2.

The highest expressing clone was used for assessing new anti-human BCMAmAbs, with untransfected CHO cells as a control. Briefly, BCMA-CHO cellswere pretreated with FACS buffer plus 5% normal mouse serum to blocknon-specific binding sites. Seven anti-BCMA mAbs—C11D5.3, C12A3.2,C13F12.1 and A7D12.2 and the commercially available anti-BCMA mAbs,C4E2.2 (Legacy Biogen), VICKY-1 (Alexis) and 335004 (R&D Systems,Inc.)—and isotype control antibodies were incubated separately withcells for 30 minutes on ice at 1 μg/ml, and washed. Biotin-conjugatedisotype control Abs were as follows: mouse IgG1, eBioscience cat. no.13-4714-85; mouse IgG2a, eBioscience, cat. no. 13-4732-85; hamster IgG1,BD Pharmingen cat. no. 553970; rat IgG2a, eBioscience, cat. No.13-4321-82. To visualize positive staining, Streptavidin-PE (MolecularProbes, Eugene, Oreg.) was added to cells for 30 minutes on ice, afterwhich cells were washed, fixed in 0.8% paraformaldehyde, and run on aFACScalibur flow cytometer (BDbiosciences, San Jose, Calif.) andanalyzed using Flowjo software (Treestar, Ashland, Oreg.). Results areshown in FIG. 1.

All seven antibodies showed specific recognition of the BCMA-expressingcells. C11D5.3 showed a slightly higher basal binding to the negativecontrol cells than the control mAb; basal binding of the other sixantibodies to the negative control cells was similar to that of thecontrol Ab.

EXAMPLE 4 Analysis of Anti-BCMA mAb Epitope Overlap by Cross-BlockingELISA

The seven anti-BCMA mAbs tested in Example 3 were then assessed in across-blocking assay to determine the presence or absence of epitopeoverlap between each antibody. Corning 96-well flat-bottom plates wereincubated overnight at 4° C. with 10 μg/ml of mouse anti-human Fc in a50 mM pH 9.6 sodium bicarbonate solution, washed, incubated with 2 μg/mlhuman BCMA-Fc (SEQ ID NO:10) at 37° C. for 1 hr, washed again, andnon-specific binding sites were blocked with blocking buffer (3% BSA inPBS) for 30 min at 37° C. Triplicate wells were then incubated with eachunconjugated anti-BCMA mAb clone in blocking buffer at a concentrationthat was ten times the concentration of the single biotin-conjugatedanti-BCMA mAb used in each individual experiment. A singlebiotin-conjugated anti-BCMA mAb clone in blocking buffer was added toall wells at a concentration pre-determined to give 80% of maximalsignal (EC80) and incubated for 1 hr at 37° C., after which wells werewashed and incubated with a streptavidin-HRP solution for 30 min, 37°C., washed, and incubated with substrate, TMB, to visualize positivereactivity. Enzymatic reactivity was stopped by adding 2N sulfuric acid,and the absorbance at 450 nm was measured using a plate reader.

For each species of biotin-conjugated antibody, control readings inwhich the unconjugated and conjugated antibodies were the same (exceptfor the presence/absence of conjugated biotin) were consideredbackground levels, i.e., the absorbance from this control was subtractedfrom each experimental reading for that antibody. In some cases, thisbackground subtraction resulted in slightly negative values. Although itis possible that the labeled antibody was blocked slightly moreeffectively by a different unconjugated antibody than by itself, theseslightly negative values might also result from experimental variation.Results were then expressed as a percentage of the positive controlvalue, i.e., the background-adjusted absorbance reading for thebiotin-conjugated antibody in the absence of a competing unconjugatedantibody.

Antibodies were considered to bind the same epitope or very closelyoverlapping epitopes if they each reduced the other's binding (accordingto the fraction calculated as above) to below 20% of the positivecontrol value. If they did not satisfy this condition, they wereconsidered to have at least partially distinct epitopes. Table 3 showswhich antibodies have at least partially distinct epitopes.

EXAMPLE 5 Flow Cytometric Analysis of Antibody Binding to HumanPeripheral Blood Cells

Blood was obtained from consenting healthy volunteers and peripheralblood mononuclear cells (PBMCs) were enriched by centrifugation throughFicoll-Paque (GE Healthcare, UK) according to the manufacturer'srecommendations. PBMCs were washed extensively in PBS prior to use.Cells were pretreated with FACS buffer containing 5% normal mouse serumto block non-specific binding sites. The following fluorphore-conjugatedmonoclonal antibodies directed against specific B cell and plasma cellmarkers were used: anti-CD19-PE-Cy5, anti-IgD-FITC, anti-CD27-APC,anti-CD38-PE-Cy7 (BD Biosciences, San Jose, Calif.). Streptavidin-PE(Molecular Probes, Eugene, Oreg.) was used to visualizebiotin-conjugated anti-BCMA mAbs (10 μg/ml). Binding of an isotypecontrol mAb as in Example 3 was also measured.

None of the anti-BCMA mAbs stained naïve B cells from healthy volunteers(FIG. 2E), while all stained plasma cells, although with varyingintensities (FIG. 2A). Only clone A7D12.2 stained a proportion of allthree memory B cell subsets (FIG. 2B-D).

EXAMPLE 6 Comparison of Antibody Binding to B Cells of Healthy andSLE-Afflicted Individuals

Blood was obtained from consenting healthy volunteers and SLE patientsand processed as in Example 5. FIG. 3 shows a comparison between samplesfrom a healthy volunteer and a representative SLE patient. AntibodyA7D12.2 bound to plasma cells from both healthy volunteers and SLEpatients (FIG. 3A). In the SLE samples but not the healthy samples, theA7D12.2 antibody bound naïve B cells (FIG. 3E). Binding of the A7D12.2antibody to memory B cells (FIG. 3B-D), particularly double negativememory B cells (FIG. 3D), was increased in SLE samples.

EXAMPLE 7 Generation of Cell Lines Producing Chimeric Anti-BCMA mAbs

CHO-DG44-I, a dhfr-deficient, insulin-independent Chinese hamster ovarycell line, was used to construct anti-BCMA wild type cell lines. Thehost cells were cultured in CHO-S-SFM II medium with nucleosides priorto transfection.

Chimeric antibodies were produced by transfecting cells with expressionplasmids encoding the mature heavy and light chain sequences listed inTable 4.

TABLE 4 Mature heavy and light chain sequences of chimeric anti-BCMAantibodies Mature heavy Chimeric antibody chain sequence Mature lightchain sequence chA7D12.2 SEQ ID NO: 13 SEQ ID NO: 14 chC11D5.3 SEQ IDNO: 15 SEQ ID NO: 16 (for Example 8) SEQ ID NO: 17 (for Example 9)chC12A3.2 SEQ ID NO: 18 SEQ ID NO: 19 chC13F12.1 SEQ ID NO: 20 SEQ IDNO: 20

Chimeric anti-BCMA expression plasmids were transfected into the CHOhost cell line DG44-I using a cationic lipid (Fugene HD) method.Briefly, 1×10⁶ DG44-I cells were seeded in each of two wells of a 6-wellplate containing 3 mL of CHO-S-SFMII medium w/nucleosides per well. Fourμg of plasmid DNA (2 μg heavy chain, 2 μg light chain) was diluted into200 μL CHO-S-SFM II (Invitrogen) medium at room temperature. Sixteen μLof Fugene HD (Roche) reagent and allowed to complex with the DNA forapproximately 15 minutes. 100 μL of the complexed DNA mixture was addedto each well containing the cells. After three days, the transfectedcells were combined and transferred to a T-75 flask containing 20 mLCHO-S-SFM II medium w/o nucleosides containing 400 μg/mL geneticin(Invitrogen). Cells were monitored for viability and scaled-upaccordingly. As the cells were scaled-up they were adapted to productionmedium. Clonal cell lines were obtained by FACS sorting individual cellsfrom the stable population.

CHO-DG44-I cells stably transfected with chimeric heavy and light chainswere fermented in CHOM39 media, harvested, and the cells were removed bycentrifugation. The pH of the cleared media was adjusted prior topassing through a protein A Fast Flow column. Antibodies were elutedwith 100 mM Na-citrate buffer, pH 3.0, neutralized to pH 7.0 using 10%(v/v) of 2M glycine, pH 8.9, and the recovered antibody solution wasbuffer-exchanged to PBS (pH 7.2) using a Superdex 200 size exclusionchromatography under endotoxin-free conditions. The purified protein waskept at −80° C.

EXAMPLE 8 Anti-BCMA-Mediated Killing In Vitro

Cells and Cell Culture

JJN-3 human plasmacytoma cells (DSMZ ACC 541) were cultured in culturemedium consisting of 40% Dulbecco's MEM, 40% Iscove's MDM, 20% FBS, 100units/mL penicillin and 100 μg/mL streptomycin. U266 human plasmacytomacells (ATCC TIB 196) were cultured in culture medium consisting of RPMI1640, 15% FBS, 20 mM HEPES, 100 units/mL penicillin and 100 μg/mLstreptomycin. All cells were cultured at 37° C. in a 5% CO2 atmosphere.Peripheral blood mononuclear cells were isolated from a consented normalhealthy donor by density centrifugation through Ficoll-Paque PLUS (GEHealthcare, Uppsala, Sweden).

In Vitro Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay

Assay diluent was RPMI 1640, 1% BSA, 20 mM HEPES, 100 units/mLpenicillin and 100 μg/mL streptomycin. Human plasmacytoma cell linesJJN-3 and U266 were washed and resuspended in assay diluent to 0.4×10⁶cells per mL. 50 μL of each cell suspension was plated into a 96-well Ubottom microtiter plate in triplicates. 50 μL of serially dilutedchimeric anti-BCMA antibodies (chC12A3.2, chC13F12.1, chC11D5.3, andchA7D12.2, as described in Example 7) and control human IgG1 (HIgG1;Protos lmmunoresearch, Burlingame, Calif.) were added to wellscontaining the cell lines and incubated for 30 minutes at 37° C. For aneffector:target ratio of 25:1, 50 μL of PBMCs (500,000) were added andincubated for an additional four hours at 37° C. in a 5% CO2 atmosphere.Plates were centrifuged at 1200 rpm for 5 minutes, and 100 μL ofsupernatant was transferred to a 96-flat-bottom microwell plate. Thelevel of cell lysis was determined by measuring the amount of lactatedehydrogenase (Cytotoxicity Detection Kit (LDH), #11 644 793 001 Roche)released from lysed cells. 100 μL of LDH kit reaction mixture was addedto 100 μL of supernatant for up to 30 minutes as followed bymanufacturer instructions. Absorbance was measured at 490 nm. Controlsincluded target cells alone (spontaneous LDH release), target cellsalone with 2% Triton X-100 in assay diluent (maximum LDH release),effector cells with and without target cells, and human IgG1 isotypecontrol.

Results

The human BCMA+ plasmacytoma cell line, U266, was utilized to test theability of chimeric anti-BCMA antibodies to kill via ADCC. Humanperipheral blood mononuclear cells (PBMCs) were used as effector cells.As shown in Table 5, chimeric mAbs C12A3.2 and C13F12.1 demonstratedmarked ADCC activity relative to HIgG1 controls. Chimeric clones A7D12.2and C11D5.3 also mediated ADCC, although to a lesser degree than C12A3.2and C13F12.1.

TABLE 5 ADCC of U266 cells by anti-human BCMA mAbs % Maximal Killing mAbClone % Killing at 1 nM (mAb concentration, nM) chC12A3.2 32   35 (4)chC13F12.1 20   20 (0.4) chA7D12.2 10   24 (100) chC11D5.3 18 ≧42(≧100)¹ ¹Maximum killing determination is incomplete

ADCC assays were also performed using a second BCMA+ plasmacytoma cellline, JJN-3, as the target cells. As shown in Table 6, chimeric C12A3.2,C13F12.1, and A7D12.2 also mediated ADCC of JJN-3 cells.

TABLE 6 ADCC of JJN-3 cells by anti-human BCMA mAbs % Maximal KillingmAb Clone % Killing at 1 nM (mAb concentration, nM) chC12A3.2 5   14(100) chC13F12.1 3 ≧14 (≧100)¹ chA7D12.2 20   37 (100) ¹Maximum killingdetermination is incomplete

Using enriched human NK cells as effector cells did not result inimproved killing for chC12A3.2, chC13F12.1 or chA7D12.2. Rather, percentkilling by NKs was diminished relative to PBMCs, indicating that NKcells are not the effector cells for chC12A3.2, chC13F12.1, or chA7D12.2in the ADCC assays with U266 and JJN3 as target cells (data not shown).Generation of afucosyl variants of chC12A3.2, chC13F12.1, or chA7D12.2did not improve activity of these mAbs (data not shown).

EXAMPLE 9 Anti-BCMA-Mediated Cell Depletion In Vivo

Establishment of Humanized (HSC/NSG) Mice

HSC/NSG mice provide a useful tool for testing in vivo biologic reagentsspecific for human protein targets since their immune system consists offunctioning human cell types (Brehm et al. (2010), Clin Immunol [Epubahead of print]). Humanized mice were generated as previously described(Pearson et al. (2008), Curr Protoc Immunol Chapter 15: Unit 15.21.PMID: 18491294). NOD/SCID/common gamma chain-deficient mice (NSG mice)were purchased from Jackson Laboratories (Bar Harbor, Me.). Mice weremaintained and bred under specific-pathogen-free conditions in isolatorcages. Breeding pairs were established and dams were monitored forpregnancy and delivery. Pups ranging in age from 2-6 days old were usedto create humanized mice. Pups were lightly irradiated, receiving a doseof 1 Gy (100 rad) from a 137 Cs irradiator. Pups were immediatelyinjected intra-orbitally with approximately 50,000 CD34+CD3-human stemcells (HSC) derived from umbilical cord blood (All Cell LLC, purchasedfrom StemCell Technologies Inc., Vancouver, BC, Canada), then returnedto the dam. Pups were weaned at 21 days and caged with littermatesaccording to gender. Starting at 3 months of age mice were bled via thefacial vein into heparinized tubes and the whole blood was analyzed byflow cytometry for the presence of human cells. In brief, 100 μl wholeblood was stained with a cocktail of mAbs, anti-human CD45-FITC,anti-human CD3-PE, anti-human CD19-PerCp, and anti-mouse CD45-APC (BDBiosciences, San Jose, Calif.). Mice were considered successfullyhumanized if they had at least 20% human CD45+ cells in whole blood ofwhich 10% or more were human CD3+, the remainder being human B cells andother human hematopoietic cells. Mice were occasionally bled andanalyzed to ensure that the humanization was stable, and were routinelyanalyzed 2 weeks prior to study enrollment.

Flow Cytometry

To identify plasma cells (PC) in human stem cell humanized NSG (HSC/NSG)mice, collagenase-digested spleens were subjected to flow cytometricanalysis. Cells were incubated with a cocktail of mAbs directed to humancell lineage and to human B cell markers. The panel consisted ofanti-human CD45, anti-human CD19, anti-human CD27, anti-human IgD, andanti-human CD38. Two markers used to exclude specific cell populationswere anti-mouse CD45 and anti-human CD3. Cells identified as PC werehuman CD45+, human CD19+, human CD3−, human CD27+, human IgD− and humanCD38bright. To identify BCMA+ cells the biotin-conjugated anti-humanBCMA mAbs, C12A3.2 and A7D12.2, were used.

In Vivo Cell Depletion Assay

5-6 month-old HSC/NSG mice received chimeric anti-BCMA mAb i.p. HumanIgG1 with no known reactivity (Protos lmmunoresearch) was used as anegative control. Blood was collected to prepare serum for analysis ofhuman Ig isoform levels. At the study terminus the spleen was harvested,a single cell suspension was prepared, RBCs were lysed and cells werewashed 3× with PBS/5% FCS and the cell number was determined. Cells wereassessed by flow cytometry for T lineage cells, B lineage cells, andplasma cells.

Assessment of Serum Human Ig Isotypes

Serum levels of human IgM and IgG were determined using an ELISA format(Bethyl Laboratories Inc., Montgomery, Tex.) and a human immunoglobulinisotyping kit (Millipore, Billerica, Mass.), respectively, according tothe manufacturer's protocol.

Results

Analysis of HSC/NSG mice aged 5-6 months revealed that among the diversecell subsets assessed BCMA+ cells were only found in the B cell lineage.Within the B cell lineage, BCMA was found only on splenic PCs (humanCD19+, human CD27+, IgD−, CD38 bright) (FIG. 4), and not on naïve Bcells (human CD19+, human CD27−, IgD+), unswitched memory B cells (humanCD19+, human CD27+, human IgD+), or switched memory B cells (humanCD19+, human CD27+, human IgD−) (data not shown).

To assess the ability of chimeric anti-human BCMA mAbs to deplete humanPC, HSC/NSG mice received various amounts of chimeric anti-human BCMAclones chAC11D5.3, chC12A3.2, chC13F12.1, and chA7D12.2 (Example 7)twice weekly i.p. for 2 weeks, after which the presence of splenic PCswas determined. Splenic PCs from the HIgG1-treated control group wereanalyzed for expression of BCMA using the A7D12.2 and C12A3.2 clones andwere confirmed to express cell surface BCMA (data not shown). PCs wereidentified using the flow cytometric parameters described above, andtotal cell numbers were determined from the flow cytometric dot plots(calculated as a percentage of the total human cell number). The numbersof naïve human B cells, unswitched memory human B cells, and switchedmemory human B cells were also determined.

Treatment with chC12A3.2 (N=5) resulted in a statistically significantdecline, 93% and 95%, in the number of splenic PCs at the 200 μg and 20μg dose levels, respectively, when compared with control HIgG-treatedmice (N=5) while the 2 82 g dose also showed a marked decline (32%),although it was not statistically significant. Treatment with chC13F12.1(N=5) resulted in a statistically significant decline, 88% and 51%, inthe number of splenic PCs at the 200 μg and 20 μg dose levels,respectively (FIG. 5). No impact on the number of other B cell subsetsor T cells was observed with chC12A3.2 and chC13F12.1 (data not shown).

Treatment with 200 μg of chC11D5.1 (N=2) or chA7D12.2 (N=1) resulted inan 89% and 97% reduction, respectively, in human PCs within the spleenwhen compared to HIgG-treated control mice (N=5) (FIG. 6). Treatmentwith chA7D12.1 also resulted in a 2.6-fold decline in the number ofsplenic human switched memory B cells when compared to HIgG-treated mice(575 vs. 217 PCs/10⁵ HuCD45⁺ cells, for HIgG and chA7D12.1,respectively). Although BCMA could not be detected on the surface ofswitched memory B cells in untreated humanized mice, it appears thatwhile the level of BCMA was below the limit of detection by flowcytometry, it was sufficient to result in Ab-mediated killing.

To determine the impact of human PC depletion on serum human Ig levelsin the HSC/NSG mice described above, human Ig subsets were analyzed. Thechimeric anti-BCMA mAbs used in these studies were of the human IgG1isoform, therefore human IgG1 levels could not be accurately evaluated.In some experimental cohorts, control-treated mice had very little andvariable amounts of isotypes IgG1, IgG2, IgA and IgE (data not shown).As shown in Table 7, both chC12A3.2 and chC13F12.1 resulted in markedreductions in serum human IgM, especially at the higher dose levels.Chimeric C12A3.2 resulted in a 63%, 62% and 30% reduction in IgM for the200, 20 and 2 μg dose levels, respectively. Chimeric C13F12.1 resultedin a 52%, 42% and 32% reduction in IgM for the 200, 20 and 2 μg doselevels, respectively.

TABLE 7 Serum human IgM levels (μg/mL) Treatment Treatment chC12A3.2chC13F12.1 Human IgG1 dose (μg) Mean SD¹ Mean SD Mean SD 200 70.2² 37.491.1³ 26.5 190.6 67.3 20 73.0² 32.2 110.3⁴ 47.3 ND⁵ 2 134.3 60.1 128.9112.4 ND ¹SD = standard deviation; ²p = 0.008; ³p = 0.02; ⁴p = 0.05; ⁵ND= not done

In a separate experiment, mice received chC12A3.2 (N=14) or HIgG1control (N=9), and the control mice had readily detectable IgG2 and IgG3isotypes as well as IgM. Chimeric C12A3.2-treated mice exhibited asignificant depletion of splenic plasma cells, similar to that seen inFIG. 5 (data not shown). As shown in Table 8, mice that receivedchC12A3.2 exhibited significantly reduced serum IgG2 and IgM levels whencompared with HIgG-treated control mice. Chimeric C12A3.2-treated micealso had a marked reduction in serum IgG3, although the difference didnot reach statistical significance when compared to control mice (Table8).

TABLE 8 Serum human immunoglobulin levels Serum Immunoglobulin (μg/mL)IgG2 IgG3 IgM Treatment Mean SE¹ Mean SE Mean SE chC12A3.2 5.4² 1.9 1.10.7 40.5³ 12.2 HIgG 19.2 7.6 6.9 4.1 114.3 19.1 ¹SE = standard error; ²p= 0.05; ³p = 0.003

The embodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications andpatents cited in this disclosure are incorporated by reference in theirentirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to be obtainedby the present invention. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated antibody that binds to the polypeptide of SEQ ID NO:9,wherein the antibody comprises at least one CDR chosen from CDR1, CDR2,or CDR3 of a protein sequence selected from the group consisting of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.
 2. The antibody ofclaim 1, wherein the antibody comprises at least two CDRs chosen fromCDR1, CDR2, or CDR3 of a protein sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. 3.The antibody of claim 1, wherein the antibody comprises the CDRs atCDR1, CDR2, and CDR3 of a protein sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. 4.The antibody of claim 1, wherein the at least one CDR chosen from CDR1,CDR2, or CDR3 is from SEQ ID NO: 5, and the antibody further comprises avariable domain comprising at least one of a CDR1 region identical toamino acids 24-38 of SEQ ID NO: 6, a CDR2 region identical to aminoacids 54-60 of SEQ ID NO: 6, or a CDR3 region identical to amino acids93-101 of SEQ ID NO:
 6. 5. The antibody of claim 1, wherein the heavychain variable domain comprises SEQ ID NO:5 and the light chain variabledomain comprises SEQ ID NO:6.
 6. An isolated antibody that binds thesame epitope as the antibody of claim
 5. 7. An isolated antibody thatbinds to the polypeptide of SEQ ID NO:9, wherein the antibody comprises:a) a variable domain comprising CDR1, CDR2, and CDR3 regions identicalto the CDR1, CDR2, and CDR3 of a sequence which is chosen from SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7; or b) a variant of thevariable domain of part (a) that is otherwise identical to said variabledomain except for up to a total of 10 amino acid substitutions in saidCDR regions.
 8. The antibody of claim 7, wherein the sequence is SEQ IDNO:5 and the antibody further comprises: c) a variable domain comprisinga CDR1 region identical to amino acids 24-38 of SEQ ID NO:6, a CDR2region identical to amino acids 54-60 of SEQ ID NO:6, and a CDR3 regionidentical to amino acids 93-101 of SEQ ID NO:6; or d) a variant of thevariable domain of part (c) that is otherwise identical to said variabledomain except for up to a total of 10 amino acid substitutions in saidCDR regions.
 9. An isolated antibody that binds to the polypeptide ofSEQ ID NO:9, wherein the antibody comprises at least one CDR chosen fromCDR1, CDR2, or CDR3 of a protein sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:6, and SEQ ID NO:8.10. The antibody of claim 9, wherein the antibody comprises at least twoCDRs chosen from CDR1, CDR2, or CDR3 of a protein sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:6, and SEQID NO:8.
 11. The antibody of claim 10, wherein the antibody comprisesthe CDRs at CDR1, CDR2, and CDR3 of a protein sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:6, or SEQ IDNO:8.
 12. The antibody of claim 1, wherein the antibody is a chimeric,humanized, or single chain antibody.
 13. A hybridoma that produces theantibody of claim
 1. 14. A pharmaceutical composition comprising theantibody of claim 1 and a pharmaceutically acceptable carrier.
 15. Apolypeptide that binds to SEQ ID NO:9 and comprises the antigen bindingportion, Fab fragment, or F(ab′)2 fragment of the antibody of claim 1.16. A hybridoma that produces the antigen binding portion, Fab fragment,or F(ab′)2 fragment of claim
 15. 17. A pharmaceutical compositioncomprising the antigen binding portion, Fab fragment, or F(ab′)2fragment of claim 15 and a pharmaceutically acceptable carrier.
 18. Amethod of depleting plasma cells, comprising administering the antibodyof claim
 1. 19. A method of treating a B cell-related disorder,comprising administering the antibody of claim
 1. 20. The method ofclaim 19, wherein the B-cell related disorder is plasmacytoma, Hodgkins'lymphoma, follicular lymphomas, small non-cleaved cell lymphomas,endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zonelymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodalmonocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma,large cell lymphoma, diffuse mixed cell lymphoma, immunoblasticlymphoma, primary mediastinal B cell lymphoma, pulmonary B cellangiocentric lymphoma, small lymphocytic lymphoma, B cell proliferationsof uncertain malignant potential, lymphomatoid granulomatosis,post-transplant lymphoproliferative disorder, an immunoregulatorydisorder, rheumatoid arthritis, myasthenia gravis, idiopathicthrombocytopenia purpura, anti-phospholipid syndrome, Chagas' disease,Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa,Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis,anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture'sdisease, Kawasaki disease, autoimmune hemolytic anemia, and rapidlyprogressive glomerulonephritis, heavy-chain disease, primary orimmunocyte-associated amyloidosis, or monoclonal gammopathy ofundetermined significance.
 21. The method of claim 20, wherein the Bcell-related disorder is a B cell malignancy.
 22. The method of claim20, wherein the B cell-related disorder is a plasma cell malignancy. 23.The method of claim 22, wherein the plasma cell malignancy is multiplemyeloma.
 24. The method of claim 20, wherein the B cell-related disorderis an autoimmune disease.
 25. The method of claim 24, wherein theautoimmune disease is systemic lupus erythematosus.
 26. The method ofclaim 20, wherein the B-cell related disorder is rheumatoid arthritis.27. The method of claim 20, wherein the the B-cell related disorder isidiopathic thrombocytopenia purpura, or myasthenia gravis, or autoimmunehemolytic anemia.
 28. The method of claim 18, further comprisingadministering RITUXAN™.
 29. A method of reducing the level of at leastone autoantibody, comprising administering the antibody of claim
 1. 30.An isolated antibody that binds to the polypeptide of SEQ ID NO:9,wherein the antibody comprises a variable domain comprising CDR1, CDR2,and CDR3 regions that are together at least 95% identical to the CDR1,CDR2, and CDR3 regions of a sequence which is chosen from SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.
 31. The antibody of claim 30,wherein the sequence is SEQ ID NO:5 and the antibody further comprises avariable domain comprising CDR1, CDR2, and CDR3 regions that aretogether at least 95% identical to the CDR1, CDR2, and CDR3 regions ofSEQ ID NO:6.